Abstract:

The present invention provides compositions and methods for protecting
cells and tissues from damage associated with therapeutic treatments of
cancers and other diseases and conditions where reactive oxygen species
are produced. The present invention also provides compositions useful as
research reagents.

7. A method for protecting cells from the toxic effects of free radical
generating therapies comprising:a) providing a subject with a condition
being treated with therapies that are toxic to normal cells and disease
cells,b) co-administering to said subject: a) said toxic therapy and, b)
a therapeutic agent, that through metabolism in said subject, causes
accumulation of a chemoprotectant compound in said normal cells at a
higher concentration than in said disease cells.

Description:

[0001]The present application claims priority to U.S. Provisional Patent
Application Ser. No. 60/920,176, filed Mar. 27, 2007, the disclosure of
which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002]The present invention provides compositions and methods for
protecting cells and tissues from damage associated with therapeutic
treatments of cancers and other diseases and conditions where reactive
oxygen species are produced. The present invention also provides
compositions useful as research reagents.

BACKGROUND OF THE INVENTION

[0003]Cancers are a leading cause of death in animals and humans. The
leading cancer therapies today are surgery, radiation and chemotherapy.
In spite of advances in the field of cancer treatment, each of these
known therapies has serious side effects. For example, surgery disfigures
the patient or interferes with normal bodily functions. Chemotherapy or
radiation therapies cause patients to experience acute debilitating
symptoms including nausea, vomiting, diarrhea, hypersensitivity to light,
hair loss, etc. The side effects of these cytotoxic compounds frequently
limit the frequency and dosage at which they can be administered. The
main reason chemotherapy is so debilitating and the symptoms so severe is
that chemotherapeutic drugs are often unable to differentiate between
normal, healthy cells and the tumor cells they are designed to target.
Therefore, as they target tumor cells they also target healthy cells
thereby causing the toxic side effects to the subject receiving the
chemotherapy. As well, radiation therapy targets the whole system, not
just tumor cells, so side effects are once again severe for the subject
receiving radiation therapy.

[0004]While chemotherapeutic compounds have been found to be effective and
are in general clinical use as anti-proliferative agents, there are well
recognized drawbacks associated with their administration.
Chemotherapeutic alkylating agents have marked cytotoxic action and the
ability of these drugs to interfere with normal mitosis and cell division
can be lethal. Chemotherapeutic antimetabolites can lead to anorexia,
progressive weight loss, depression, and coma. Prolonged administration
of antimetabolites can result in serious changes in bone marrow. Both the
alkylating agents and the antimetabolities generally have a depressive
effect on the immunosuppressive system. Prolonged administration of
natural products such as vinca alkyloids can also result in bone marrow
depression. Hydroxy urea and other chemically derived chemotherapeutic
agents can lead to rapid reduction in levels of adrenocorticosteroids and
their metabolites. The administration of hormonal chemotherapeutic
compounds or radioactive isotopes is also undesirable from the viewpoint
of inflicting damage on the immunosuppressive system and thereby
disabling the body's defenses against common infections. Moreover, it is
recently reported that cognitive function is compromised upon
administration of some chemotherapeutic compounds, in particular the
administration of adriamycin in treating breast cancer. Such cognitive
dysfunction is loosely termed "chemo brain", and is marked by increased
oxidative stress and cellular apoptosis in the brain (Joshi et al., 2007,
J. Neurosci. Res. 85:497-503).

[0005]Glutathione (GSH) represents one of the most prevalent organic
molecules within the cell, with concentrations ranging from 0.1 to 15 mM.
Glutathione functions primarily as an antioxidant, reacting with toxic
species as well as serving as a cofactor for a number of protective
enzymes such as glutathione peroxidase and glutathione transferase.
Glutathione is also an important determinant of the cell's ability to
pump toxic substances, such as chemotherapeutic drug metabolites, out of
the cell. The concentration of glutathione and the extent of glutathione
oxidation are thought to be a key determinant of cells undergoing
programmed cell death (apoptosis) in response to chemotherapy or
radiation therapy.

[0006]Several sulfhydryl containing compounds have been developed to
protect normal tissues from the toxic effects of either chemotherapy or
radiation therapy. For example, glutathione has been utilized in clinical
trials to protect against the toxic effects of chemotherapy. Cascinu et
al. (2002, J. Clin. Once. 20:3478-83) found that co-administration of
reduced glutathione could significantly reduce the neuropathy seen with
the chemotherapeutic drug oxaliplatin. However, the effect of reduced
glutathione is relatively limited in that this compound is rapidly
hydrolyzed when given intravenously. Unfortunately, systemically
administered glutathione protects tumor cells and normal cells equally
and has not been shown to improve the therapeutic index. Also, elevation
in glutathione levels is a common characteristic of tumor cells resistant
to chemotherapy (Moscow and Dixon, 1993, Cytotech. 12:155-70).

[0007]Sodium 2-mercaptoethane sulphonate (Mesna) is a thiol-producing
compound that is used in clinical oncology to prevent bladder damage from
high doses of chemotherapeutic alkylating agents (e.g., cyclophosphamide,
cisplatin, ifosfamide, carboplatin, doxorubicin and its derivatives,
mitomycin and its derivatives). Mesna (UROMITEXAN, MESNEX; U.S. Pat. Nos.
5,661,188, 6,696,483 and 6,462,017) is excreted rapidly in the urine
which limits its general utility except for bladder protection.

[0008]Amifostine (ETHYOL, WR-2721; U.S. Pat. Nos. 7,151,094, 6,841,545,
6,753,323, 6,407,278, 6,384,259, 5,994,409) was developed as a radiation
protection agent by the U.S. Walter Reed Army Institute of Research in
the 1950s. Amifostine (S-2-(3-aminopropylamino)ethylphosphorothioic acid)
is a cytoprotective adjuvant used in cancer chemotherapy involving
DNA-binding chemotherapeutic agents and is used therapeutically to reduce
the incidence of fever and infection induced by DNA-binding
chemotherapeutic agents including alkylating agents (e.g.
cyclophosphamide) and platinum-containing agents (e.g. cisplatin). It is
also used to decrease the cumulative nephrotoxicity associated with
platinum-containing agents and is indicated to reduce the incidence of
dry mouth in patients undergoing radiotherapy for head and neck cancer.
Amifostine is an organic thiophosphate prodrug that is dephosphorylated
in vivo by alkaline phosphatase (e.g., alkaline phosphatase is capable of
hydrolyzing phosphorothioates in addition to phosphoether moieties in a
variety of compounds) to the active cytoprotective thiol metabolite
(WR-1065). The selective protection of non-malignant tissues is believed
to be due to higher alkaline phosphatase activity, higher pH, and
vascular permeation of normal tissues; dephosphorylation takes place
preferentially in normal blood vessels but to a much lesser extent in
tumor vessels because tumors are more acidic and the newly formed tumor
blood vessels do not significantly express the enzyme alkaline
phosphatase. In randomized Phase III human trials, amifostine has been
shown to reduce toxicity with 1) chemotherapy and radiation therapy in
head and neck cancer (David et al., 2000, J. Clin. Once. 18:3339-45); 2)
radiation therapy in lung cancer patients (Antonadou et al., 2001, Int.
J. Rad. Once. Biol. Phys. 51:915-22); 3) myelosuppression from
carboplatin; and 4) chemotherapy and radiation therapy in rectal cancer.
Amifostine was originally indicated to reduce the cumulative renal
toxicity from cisplatin in non-small cell lung cancer. However, while
nephroprotection was observed, the fact that amifostine could protect
tumors could not be excluded. Therefore, given better treatment options
for non-small cell lung cancer, amifostine's indication for non-small
cell lung cancer was withdrawn in 2005.

[0009]As such, what are needed are novel compositions for use as
broad-spectrum chemoprotectants and radioprotectants. Such novel
compositions would not only serve as adjuvants to chemo and radiation
therapies to protect the subjects normal cells from the toxicity
associated with such therapies, but such novel compositions would also
prove useful as research reagents in the study of, for example,
chemotherapeutics and cellular biology.

SUMMARY OF THE INVENTION

[0010]The present invention provides compositions and methods for
protecting cells and tissues from damage associated with therapeutic
treatments of cancers and other diseases and conditions where reactive
oxygen species are produced. The present invention also provides
compositions useful as research reagents.

[0011]In one embodiment, the compositions of the present invention are
used in conjunction with cytotoxic chemotherapy and/or radiation therapy
in the treatment of subjects, and are broadly applicable to such
treatment regimens. It is contemplated that by decreasing toxicity to
normal cells, the compositions thereby allow for the escalation (e.g.,
high dose, prolonged treatment, use of drugs otherwise considered too
toxic, etc.) of chemotherapy or radiation dosing, resulting in more
effective treatments. Likewise, the compounds find use in conjunction
with existing therapeutic protocols to reduce toxicity and the associated
underlying sign, symptoms, and side effects.

[0012]In some embodiments, the compositions and methods of the present
invention find utility in protecting normal cells from toxicity due to
treatment regimens associated with cellular toxicity due to, for example,
AIDS, anti-fungal therapy, antibacterial therapy, and intravenous
contrast agents. The compositions and methods can also be used to treat
disorders that are induced by aging and metabolic disorders, including,
but not limited to diabetes.

[0013]The present invention provides compositions and methods for the
treatment of a wide variety of metabolic processes and disorders wherein
free radicals, and therefore cell damage or apoptosis, can occur. The
methods of the present invention are also suitable for the treatment of
disorders relating to basal metabolism such as heat production of an
individual at the lowest level of cell chemistry in the waking state, or
the minimal amount of cell activity associated with the continuous
organic functions of respiration, circulation and secretion; carbohydrate
metabolism such as the changes that carbohydrates undergo in the tissues,
including oxidation, breakdown, and synthesis; electrolyte metabolism
such as the changes which the various essential minerals, sodium,
potassium, calcium magnesium, etc. undergo in the fluids and tissues of
the body; fat metabolism such as the chemical changes, oxidation,
decomposition, and synthesis, that fats undergo in the tissues; protein
metabolism such as the chemical changes, decompositions, and synthesis
that protein undergoes in the tissues; and respiratory metabolism such as
the exchange of respiratory gases in the lungs and the oxidation of
foodstuffs in the tissues with the production of carbon dioxide and
water.

[0014]In one embodiment, the present invention provides compositions and
methods for protecting tissues and cells from damage caused by any
therapy to a subject that is toxic to normal cells (e.g., non-diseased
cells such as non-cancerous cells), for example chemotherapy or radiation
therapy. In some embodiments, the present invention inhibits or decreases
apoptosis in normal cells and tissues due to therapies such as, for
example, chemotherapy and radiation therapy.

[0015]In one embodiment, the compositions of the present invention provide
research reagents for the scientific community for use in experimental
methods. In some embodiments, the compositions are used in in vitro
assays. In some embodiments, the compositions are used in in vivo assays.

[0016]The present invention relates, in part, to compositions and methods
for treating cellular toxicities associated with the administration to a
subject of one or more therapeutic agents, which comprise administering a
therapeutically effective amount of one or more compositions of the
present invention, or pharmaceutically acceptable salts thereof, to the
subject receiving said one or more therapeutic agents.

[0017]In some embodiments, the therapeutic agent utilized is one that
permits regioselective increase of the concentration of a natural,
non-toxic, protective material in healthy tissue. Preferably, said
compound is not increased, or increased to a lesser extent, in a cell
that is targeted for killing (e.g., a cancer cell). For example, in some
embodiments, the therapeutic agent provides a regioselective increase in
the concentration of glutathione in healthy tissue. Examples of
therapeutic agents that produce this effect are shown in Formula I, II,
and III. The present invention is not limited to these specific
compounds. In some embodiments, the therapeutic agent is a protected
glutathione molecule that can undergo a selective deprotection process
(e.g., a two-step deprotection process) that locally increases the
concentration of deprotected glutathione in cells of interest (e.g.,
healthy tissue). In some embodiments designed to provide glutathione to
cells of interest, the therapeutic agent involves carboxyl group
protection. In some embodiments, one of the carboxyl groups of
glutathione is protected. In some embodiments, both carboxyl groups of
glutathione are protected. In some embodiments, a phosphorothioate
derivative of glutathione is provided, including mono- and di-ethyl
esters thereof. In some embodiments, one or more methyl or ethyl groups
are used to protect one or more carboxyl groups of a glutathione molecule
(see e.g., Formula I, II, and III). In some embodiments, any protecting
group that can be cleaved (e.g., by a cellular esterase) is employed.
Preferably, the product of the cleavage is minimally toxic or non-toxic.
Preferably, the product is natural glutathione or a functionally
equivalent derivative thereof. In some embodiments, the protecting group
is a polyethylene glycol (PEG). In some embodiments, the protecting group
is any organic moiety that facilitates membrane permeability, including
short peptide or other materials useful for facilitating drug delivery.

[0018]The present invention is not limited to the use of glutathione as a
protective agent. In some embodiments, the therapeutic agent is any
protective agent that, alone or in combination with other agents, when
modified in vivo in a regioselective manner, provides a free-radical
scavenger in the desired target cell. For example, the therapeutic agent
may comprise alpha-lipoic acid comprising a phosphate protecting group or
other protecting group (e.g., PEG) protecting the carboxyl group. A
variety of compounds may be employed that can undergo regioselective
deprotection to provide intracellular protective compounds.

[0019]In some embodiments, the therapeutic agent is provided as part of a
bioconjugate or complex. For example, in some embodiments, the
therapeutic agent is provided in, on, or with a nanoparticle, liposome,
micelle, dendrimer, or other biocompatible material or biopolymer (e.g.,
carbohydrate) useful as a drug carrier.

[0020]In one embodiment, the present invention relates to compositions and
methods for treating cellular toxicities associated with administration
of a chemotherapeutic agent or other toxic agent wherein a composition
comprising Formula I, II, or III, other compounds described herein, or
salts, metabolites, functional derivatives, functional analogues, esters
and pro-drugs thereof, are administered prior to, with, and/or after
administration of the chemotherapeutic agent, or alternatively, at the
first indication of toxicity caused by the chemotherapeutic agent(s).
Toxicity is caused by, for example, those compounds as listed in Table 1.

[0021]The present invention further relates to methods for treating
cellular toxicities associated with the administration of therapeutic
agents by administering a composition comprising Formula I, II, or III,
other compounds described herein, or salts, metabolites, functional
derivatives, functional analogues, esters and pro-drugs thereof after
clinical appearance of toxicities following therapeutic treatment. In
some embodiments, the invention relates to methods of treating toxicities
associated with the exposure of a subject to radiation therapy, which
comprise administering to the subject a therapeutically effective amount
of one or more of the compositions as described herein, or a
pharmaceutically acceptable salt thereof, concurrent with, or after the
occurrence of, radiation therapy. In one embodiment, the present
invention relates to compositions and methods for treating cellular
toxicities associated with administration of a radiation therapy regimen
wherein a composition comprising Formula I, II, or III, other compounds
described herein, or salts, metabolites, functional derivatives,
functional analogues, esters and pro-drugs thereof, are administered
prior to, with, and/or after administration of the radiation therapy, or
alternatively, at the first indication of toxicity caused by the
radiation therapy.

[0022]In one embodiment, the present invention provides a composition
comprising Formula I. In some embodiments, Formula I comprises R1
and R2 groups that are each independently ethyl or methyl groups. In
some embodiments, the present invention provides a composition comprising
Formula I wherein n is 2. In some embodiments, the present invention
provides a composition comprising Formula I wherein R1 and R2
groups that are ethyl groups and n is 2. In some embodiments, Formula I
comprises a monosodium salt of the phosphorothioate group.

[0024]In one embodiment, the present invention provides a method for
protecting cells from the toxic effects of free radical generating
therapies comprising providing a subject with a conditions being treated
with therapies that are toxic to normal cells and disease cells, and
co-administering to said subject a composition comprising Formula I and a
therapy that is toxic to said normal cells and disease cells.

[0025]In one embodiment, the present invention provides a method of
treating subjects with cancer comprising providing a subject with cancer
and co-administering to said subject a treatment regimen comprising
Formula I and a chemotherapy drug and/or radiation therapy.

DESCRIPTION OF THE FIGURES

[0026]FIG. 1 depicts a synthesis method of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt, an embodiment of the invention, as
described in Example 1.

DEFINITIONS

[0027]As used herein, the term "subject" refers to any animal (e.g., a
mammal), including, but not limited to, humans, non-human primates,
rodents, and the like, which is to be the recipient of a particular
treatment. Typically, the terms "subject" and "patient" are used
interchangeably herein in reference to a human subject.

[0029]As used herein, the term "cell culture" refers to any in vitro
culture of cells. Included within this term are continuous cell lines
(e.g., with an immortal phenotype), primary cell cultures, transformed
cell lines, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro.

[0030]As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an artificial
environment. In vitro environments can consist of, but are not limited
to, test tubes and cell culture. The term "in vivo" refers to the natural
environment (e.g., an animal or a cell) and to processes or reaction that
occur within a natural environment.

[0031]As used herein, the term "co-administration" refers to the
administration of both a composition of the present invention with
another type of therapy, for example chemotherapy or radiation therapy.
Co-administration can be at the same time in the same administrative form
(e.g., injection, pill, liquid), or co-administration can be two
compositions given at the same time, but not in the same administrative
form.

[0032]As used herein, the term "reactive oxygen species" refers to highly
reactive chemicals, containing oxygen, that react easily with other
molecules, resulting in potentially damaging modifications. Reactive
oxygen species include, for example, oxygen ions, free radicals and
peroxides both inorganic and organic such as hydrogen peroxide,
superoxide, hydroxyl radical, lipid hydroperoxidase and singlet oxygen.
They are generally very small molecules and are highly reactive due to
the presence of unpaired valence shell electrons.

[0033]As used herein, "toxic effects" refers to damaging modifications to
cells and tissues caused by reactive oxygen species. For example, a toxic
effect of a reactive oxygen species is a cell that is modified to undergo
apoptosis.

[0034]As used herein, "free radical generating therapies" refers to drugs,
chemicals, small molecules, peptides, radiation, and other such therapies
that are applied to subjects, either alone or in combination, to treat a
disorder or disease, wherein such a therapy results in the generation of
free radicals in both non-diseased and diseased cells and tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0035]Certain illustrative embodiments of the invention are described
below. The present invention is not limited to these embodiments.

[0036]The compositions of the present invention provide novel
chemoprotectants that, when administered to a subject receiving chemo or
radiation therapy, selectively protects the subject's cells and tissues,
and not tumor tissues, from toxic therapeutic effects. Once activated,
compositions of the present invention serve, for example, as a direct
precursor to glutathione, a key regulator of apoptosis. The presence of a
phosphorothioate moiety, or other protecting moiety, in the compositions
as described herein requires cleavage by alkaline phosphatase, present in
normal cells but much less so in tumor neovasculature. Elevations of
glutathione in normal tissues render the patient less susceptible to the
toxic effects of chemotherapy and radiation therapy, whereas cancerous
cells within a tumor are not so protected.

[0037]In some embodiments, the compositions as described herein undergo
dephosphorylation (e.g., by alkaline phosphatase) in vitro under
experimental parameters or in vivo in the normal cells and tissues of a
subject. Once dephosphorylated, the composition comprises an active free
sulfhydryl (thiol, --SH) group that protects against the toxicities
associated with chemotherapy and radiation therapy by acting as a
scavenger for reactive oxygen species created by such therapies (Yuhas,
1977, in: Radiation-Drug Interactions in Cancer Management, pp. 303-352);
Yuhas, 1973, J. Natl. Cancer Inst. 50:69-78; incorporated by reference
herein in their entireties).

[0038]In one embodiment, the present invention relates to protection of
non-diseased cells and tissues by administering prior to, during, or
after, irradiation and/or chemotherapy to a tumor tissue, a
therapeutically effective amount of a composition as described herein. In
some embodiments, the administration of a composition of the present
invention is directed specifically to the non-diseased cells and tissues,
whereas the administration of the chemotherapy and/or irradiation is not
so discriminating.

[0039]In one embodiment, the compositions of the present invention include
small molecules, or analogs thereof, of the structure as seen in Formula
I:

wherein:R1 and R2 are each, separately, hydrogen, methyl, or
ethyl; andn is an integer from 2 to 10.

[0040]In one embodiment, the present invention provides salts, solvates
and hydrates of the compounds as described herein. An example of an
acceptable salt is found in Formula II:

wherein:R1 and R2 are each, separately, hydrogen, methyl, or
ethyl; andn is an integer from 2 to 10.

[0041]In some embodiments, a further example of a salt composition
suitable for use as a composition in the methods of the present
application is found in Formula III:

wherein n is an integer from 2 to 10.

[0042]In some embodiments, two or more therapeutic molecules of interest
are provided in a single therapeutic agent as a single molecule, such
that the two or more therapeutic molecules of interest are generated
intracellularly. One or more of the constituents may also be selected to
increase molecule stability, cell permeability, or other desired
properties. For example, in one embodiment, the compositions of the
present invention include small molecules, or analogs thereof, of the
structure as seen in Formula IV:

The compound of Formula IV is metabolized to provide both glutathione and
lipoic acid to a cell, each providing protection against toxic agents or
conditions. Such a molecule undergoes, for example, cleavage of the thiol
protecting phosphate by alkaline phosphatase. It is contemplated that the
nonpolar molecule is readily cell permeable. Esterase cleavage of the
conjugate and liberation of the glutathione molecule and alpha-lipoic
acid provide intracellular protection.

[0043]Therapeutic agents can also be provided as dimers or other multimers
of protective molecules. For example, in some embodiments, the
therapeutic agent comprises a molecule as seen in Formula V, a protected
dimer of glutathione:

[0044]In some embodiments, compositions of the present invention are
co-administered with chemotherapy and/or anticancer therapy and/or
radiation therapy and another chemoprotectant compound (e.g., amifostine,
mesna). In some embodiments, the administration of a composition of the
present invention is directed specifically to the non-diseased cells and
tissues, whereas the administration of the chemotherapy and/or
irradiation is not so discriminating.

[0045]For example, Table 1 lists compounds for co-administration with a
composition of the present invention.

Numerous other examples of chemotherapeutic compounds and anticancer
therapies suitable for co-administration with the disclosed compositions
are known to those skilled in the art.

[0046]In some embodiments, the compositions of the present invention are
especially useful when co-administered with an anti-cancer drug whose
cytotoxicity is due primarily to the production of reactive oxygen
species, for example, doxorubicin, daunorubicin, mitocyn C, etoposide,
cisplatin, arsenic tioxide, ionizing radiation and photodynamic therapy.

[0048]For a more detailed description of anticancer agents and other
therapeutic agents, those skilled in the art are referred to any number
of instructive manuals including, but not limited to, the Physician's
Desk Reference, Goodman and Gilman's "Pharmaceutical Basis of
Therapeutics" 10th Edition, Eds. Hardman et al., 2002 and later editions,
and "Biologic Therapy of Cancer, 2nd Edition, Eds. DeVita et al., 1995,
JB Lippincott Co. Publ, p. 919 and later editions, incorporated herein by
reference in their entireties.

[0049]In some embodiments, GSH levels in cells, for example both normal
and tumor cells, are reduced prior to the administration of compounds of
Formula I, II, or III. By lowering GSH levels in all cells, cancer cells
become vulnerable to therapies. However, following treatment with Formula
I, II, or III, normal cells are made substantially more resistant to the
toxic effects of the cancer therapies. Thus, in these embodiments, cancer
cells are supersensitized to therapy, while normal cells are protected.
The present invention is not limited by the nature of the compound or
treatment used to reduce GSH levels.

[0050]In one embodiment, the present invention provides for the use and
administration of 2-amino-4-(S-butylsulfonimidoyl)butanoic acid
(buthionine sulfoximine or BSO) in conjunction with the compositions of
the present invention. In some embodiments, buthionine sulfoximine
inhibits the synthesis of GSH in both non-tumor and tumor cells by
inhibiting γ-glutamulcysteine synthetase, an essential enzyme for
synthesis of GSH, and a composition of the present invention replenishes
GSH in non-tumor cells. In some embodiments, BSO is administered prior to
the administration of a composition of the present invention. In some
embodiments, BSO is administered in conjunction with a compositions of
the present invention. In some embodiments, the BSO and a composition of
the present invention are administered prior to, at the same time, or
after the administration of chemotherapeutics and/or radiotherapy to a
subject. It is contemplated that as BSO decreases the amount of GSH in
tumor and non-tumor cells, the addition of a composition of the present
invention replenishes GSH in non-tumor cells but not tumor cells, as such
the tumor cells maintain low or non-existent GSH levels throughout the
administration of chemotherapeutic drugs and/or radiotherapy. The low or
non-existent levels of GSH in tumor cells following administration of BSO
strips them of the protective effects that GSH offers tumor cells,
thereby allowing for more efficient targeting and eradication of the
tumor cells by chemo and radiation therapies. In some embodiments, the
administration of BSO and a compound of the present invention allows for
the administration of lesser amounts (potentially for longer time
periods) of chemotherapeutic drugs than normal due to the low or
non-existent levels of GSH in tumor cells, and at the same time the
non-tumor cells of a subject are less exposed to the toxic effects of the
therapy.

[0051]In some embodiments, the compositions of the present invention are
useful in preparation as adjuvants to chemo and/or anticancer therapy and
radiation therapy. The methods and techniques for preparing medicaments
comprising a composition of the present invention are well-known in the
art. Exemplary pharmaceutical formulations and routes of delivery are
described below. One of skill in the art will appreciate that any one or
more of the compounds described herein, including the many specific
embodiments, are prepared by applying standard pharmaceutical
manufacturing procedures. Such medicaments can be delivered to the
subject by using delivery methods that are well-known in the
pharmaceutical arts.

[0052]In some embodiments of the present invention, the compositions are
administered alone, while in some other embodiments, the compositions are
preferably present in a pharmaceutical formulation comprising at least
one active ingredient/agent, as defined above, together with one or more
pharmaceutically acceptable carriers and optionally other therapeutic
agents. Each carrier should be "acceptable" in the sense that it is
compatible with the other ingredients of the formulation and not
injurious to the subject.

[0053]Formulations include, for example, parenteral administration (e.g.,
subcutaneous, intramuscular, intravenous, intradermal) and site-specific
administration. In some embodiments, formulations are conveniently
presented in unit dosage form and are prepared by any method known in the
art of pharmacy. Such methods include the step of bringing into
association the active ingredient with the carrier that constitutes one
or more accessory ingredients. In general, the formulations are prepared
by uniformly and intimately bringing into association (e.g., mixing) the
active ingredient with liquid carriers or finely divided solid carriers
or both, and then if necessary shaping the product.

[0054]Formulations suitable for parenteral administration include aqueous
and non-aqueous isotonic sterile injection solutions which may contain
antioxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include suspending
agents and thickening agents, and liposomes or other microparticulate
systems which are designed to target the compound to blood components or
one or more organs. In some embodiments, the formulations are
presented/formulated in unit-dose or multi-dose sealed containers, for
example, ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile liquid
carrier, for example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from
sterile powders, granules and tablets of the kind previously described.

[0055]It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention may
include other agents conventional in the art having regard to the type of
formulation in question, for example, those suitable for oral
administration may include such further agents as sweeteners, thickeners
and flavoring agents. It also is intended that the agents, compositions
and methods of this invention be combined with other suitable
compositions and therapies.

[0056]Various delivery systems are known and can be used to administer
compositions of the present invention. Methods of delivery include, but
are not limited to, intra-arterial, intra-muscular, intravenous, and site
specific. For example, in some embodiments, it may be desirable to
administer the compositions of the invention locally to the area targeted
by chemo and/or anticancer therapies and/or radiation therapy; this may
be achieved by, for example, and not by way of limitation, local infusion
during surgery, injection, or by means of a catheter.

[0057]In some embodiments, in vivo administration of the compositions as
described herein is effected in one dose, continuously or intermittently
throughout the course of treatment. Methods of determining the most
effective means and dosage of administration are well known to those of
skill in the art and vary with, for example, the composition used for
therapy, the target cell being treated and the subject being treated.
Single or multiple administrations are carried out with the dose level
and pattern being selected by the treating physician. In some
embodiments, the compositions as described herein are delivered to the
subject prior to administration of the chemotherapeutic agent. In some
embodiments, compositions as described herein are delivered on a daily
basis (e.g., at least once, at least twice, at least three times) and
accompany the administration of radiotherapy.

[0058]Suitable dosage formulations and methods of administering the agents
are readily determined by those of skill in the art. When the
compositions described herein are co-administered with another
chemoprotective agent, the effective amount may be less than when the
agent is used alone. Ideally, the agent should be administered to achieve
peak concentrations of the active compound at the target sites for chemo
and radiation therapy. Desirable blood levels of the agent may be
maintained by a continuous infusion to provide a therapeutic amount of
the active ingredient within the target tissue.

[0059]The present invention also includes methods involving
co-administration of the compositions described herein with one or more
additional active agents. Indeed, it is a further aspect of this
invention to provide methods for enhancing prior art therapies and/or
pharmaceutical compositions by co-administering a compound of this
invention. In co-administration procedures, the agents may be
administered concurrently or sequentially. In one embodiment, the
compounds described herein are administered prior to the other active
agent(s). The pharmaceutical formulations and modes of administration may
be any of those described above. In addition, the two or more
co-administered chemical agents, biological agents or radiation may each
be administered using different modes or different formulations.

[0060]The agent or agents to be co-administered depends on the type of
condition being treated. For example, when treating cancer, the
additional agent is a chemotherapeutic agent, anticancer agent, or
radiation. The additional agents to be co-administered, such as anticance
can be any of the well-known agents in the art, including, but not
limited to, those that are currently in clinical use (see Table I for
exemplary agents). The determination of appropriate type and dosage of
radiation treatment is also within the skill in the art or can be
determined with relative ease.

[0061]Treatment of the various conditions associated with abnormal
apoptosis is generally limited by the following two major factors: (1)
the development of drug resistance and (2) the toxicity of known
therapeutic agents. In certain cancers, for example, resistance to
chemicals and radiation therapy has been shown to be associated with
inhibition of apoptosis. Some therapeutic agents have deleterious side
effects, including non-specific lymphotoxicity, renal and bone marrow
toxicity.

[0062]The compositions and methods described herein address both these
problems. Drug resistance, where increasing dosages are required to
achieve therapeutic benefit, is overcome by co-administering the
compositions described herein with the known agent. The compositions
described herein protect cells and tissues from toxic effects of
chemotherapeutic drugs and radiation therapy and, accordingly, less of
these agents are needed to achieve a therapeutic benefit. Conversely, the
protection of normal cells and tissues against the toxic effects of
anticancer therapies by co-administration of the compositions as
described herein allows for higher doses and/or longer treatment regimens
when using such therapies, thereby providing the medical practitioner
with the tools to follow a more aggressive anticancer strategy than was
otherwise deemed possible.

[0063]In some embodiments, the present invention provides methods for
using the compositions as described herein for screening for the efficacy
of such compositions in inhibiting or decreasing toxicity in cells and
tissues when such cells and tissues are administered cancer, or other,
therapies that are toxic to normal cells. In some embodiments, methods
for screening are conducted in vitro. In other embodiments, these screens
are conducted in vivo. In some embodiments, methods of the present
invention are performed in vivo in non-human animals or human subjects.
In some embodiments, the methods screen for the inhibition or decrease of
apoptosis is cells, in vitro or in vivo, when such cells, non-human
animals, or human subjects are co-administered a cancer, or other,
therapy in combination with compositions of the present invention. In
some embodiments, such methods define efficacy of the compositions as
described herein for use in decreasing or inhibiting the toxic effects of
therapies by comparing results from a screen with a composition of the
present invention to a screen performed without said composition (e.g.,
control experiment). Toxic effects of therapies on cells includes
cellular death by apoptosis as a result of the therapy. A composition of
the present invention that is efficacious in inhibiting or decreasing the
toxic effects of therapies is one that inhibits or decreases cellular
apoptosis in normal, non-diseased cells when toxic therapies are
administered. A skilled artisan will understand methods for determining
cellular apoptosis. These methods include, but are not limited to,
measuring apoptotic indicator enzymes such as caspase 3/7, 8 or 9,
TdT-mediated dUTP Nick-End Labeling (TUNEL) assays, and apoptosis related
antibodies (e.g., anti-PARP, anti-caspase 3, etc.). Detection methods
utilized with apoptotic assays include fluorometric, luminescent, and
calorimetric.

[0064]In some embodiments, such in vivo uses are, for example, performed
by taking a subject (e.g., human or non-human animal) with cancer and
co-administering a therapy regimen in conjunction with a composition of
the present invention, and comparing the outcome of such an
administration with a subject that received the same therapy regimen
without co-administration of a composition of the present invention.

[0065]In some embodiments, such in vitro uses are, for example, performed
in tissue culture dishes with primary or immortalized tissue culture
cells (e.g., HeLa, HEK293, CHO, 3T3, etc.) or tissue explants. In such in
vitro uses, a composition of the present invention is co-administered
with a therapy regimen known to be toxic to normal cells, the results
being compared with results from tissue culture cells or explants that
receive the same therapy regimen without a composition of the present
invention.

EXPERIMENTAL

[0066]The following examples are provided in order to demonstrate and
further illustrate certain preferred embodiments and aspects of the
present invention and are not to be construed as limiting the scope
thereof. In the experimental disclosure which follows, the following
abbreviations apply: equiv (equivalents); M (Molar); N (Normal); mol
(moles); mmol (millimoles); g (grams); L (liters); ml (milliliters);
0° C. (degrees Centigrade); min. (minutes); % (percent); psi
(pounds per square inch).

Example 1

Preparation of PBS1000

Synthesis of 2-benzyloxycarbonylamino-4-carbamoyl-butyric acid (1)

[0067]Glutamine (36.5 g, 0.25 mol) was stirred with 1M sodium bicarbonate
(750 ml) and toluene (200 ml). Benzyl chloroformate (50 ml, 59.75 g, 0.35
mol, 1.4 equiv.) was added drop-wise over 20 min. and the resulting
mixture was stirred under nitrogen at room temperature overnight. Ethyl
acetate (400 ml) was added and phases were separated. The organic phase
was extracted with water (50 ml) and discarded. The aqueous phase was
acidified with 6N hydrochloric acid and extracted with ethyl acetate
(2×600 ml). The combined extracts were washed with water (100 ml)
and stripped. The residue was dried in a vacuum oven (50° C.) to
produce (1) (64 g, 91.4%).

[0068]A mixture of acid (1) (64 g, 0.228 mol), dimeththylformamide (210
ml) and sodium bicarbonate (111 g, 1.32 mol, 5.8 equiv.) was stirred at
room temperature for 30 min. Ethyl iodide (34 ml, 66.3 g, 0.425 mol, 1.86
equiv.) was added and stirring was continued overnight under nitrogen.
The reaction mixture was slowly diluted with water to 1 L and stirred for
40 min. The solid was collected by filtration, washed well with water and
partitioned between ethyl acetate (8 L) and water (3 L). Phases were
separated and the aqueous phase was extracted with ethyl acetate (2.5 L).
The combined organic extracts were washed with water (1 L), dried over
sodium sulfate, stripped and dried in a vacuum oven (50° C.) to
produce (2) (47 g, 66.7%).

[0069]A suspension of the amide (2) (38 g, 0.1233 mol) in anhydrous
acetonitrile (400 ml) was stirred at reflux under nitrogen and t-butyl
nitrite (35 ml, 3.17 equiv.) was added quickly. The reflux was continued
for 2 hrs. After cooling, the solvent was removed in a rotary evaporator.
The residue was taken in water (250 ml) and ethyl acetate (500 ml) and
the biphasic mixture was stirred well while solid sodium bicarbonate was
slowly added to pH=7.5. Phases were separated and the organic phase was
washed with 10% sodium bicarbonate (200 ml). The combined aqueous
extracts were washed with ethyl acetate (300 ml), made acidic with 6N
hydrochloric acid and extracted with ethyl acetate (2×300 ml). The
combined extracts were washed with water (150 ml), dried over sodium
sulfate, stripped and the residue was dried in a vacuum oven (50°
C.) to produce (3) (24.6 g, 64.7%).

[0071]A solution of (4) (11.4 g, 26.86 mmol) in ethanol (225 ml)
containing 1.2 g 20% palladium on activated carbon (50% wet) was
hydrogenated at 30 psi for 3 hrs. The catalyst was removed by filtration
and the solution was washed with ethanol. The solvent was removed in a
rotary evaporator. The residue was dried in a vacuum oven (50° C.)
to produce (5) (5.86 g, 75%).

[0072]A solution of alcohol (5) (0.29 g, 1 mmol) in dichloromethane (10
ml) was treated with thionyl chloride (1 g) and stirred at room
temperature under nitrogen overnight. The solvent was removed on a rotary
evaporator (bath temperature below 28° C.). Dichloromethane (10
ml) was added and stripped under the same conditions twice. The solid
residue was taken in water (8 ml) and washed with MTBE (2×15 ml).
The resulting aqueous solution contains pure (6) (LCMS) and was used as
such in the next step.

[0073]A solution of trisodium thiophosphate (0.4 g) in water (6 ml) was
stirred at room temperature under nitrogen and the solution of (6)
prepared above was added all at once. The reaction mixture was stirred at
room temperature under nitrogen overnight. The pH was carefully adjusted
to 8.0 with acetic acid and the resulting solution was run through a
reverse phase column (P18) using water as the eluent. Fractions were
checked by LCMS and those containing the product were evaporated to
dryness (oil pump vacuum, bath temperature below 25° C.) to
produce 47 mg of (7). LCMS (M=386), 1H NMR and 31P NMR were
used to confirm the final structure (7).

[0074]Assays were performed to verify the ability of alkaline phosphatase
to dephosphorylate compound (7) to create sulfhydryl reactive groups.
Calf intestinal alkaline phosphatase (CIAP, Sigma) was diluted in
phosphate buffered saline (PBS) to 250 units/ml, and frozen in tubes
containing 100 μl aliquots. The following solutions were prepared; 2
mM glutathione (GSH), 1.05 mM DTNB (5-5'-Dithio-bis-(2-nitrobenzoic acid;
also known as Ellman's Reagent) and 5 mM amifostine (AF; 1 mg/ml).
Alkaline phosphatase activity, and the ability of the assay to measure
reactive sulfhydryl groups in solution, were evaluated initially using
amifostine as the control composition. Absorbances were measured at
A412. An increase in absorbance is indicative of free reactive
sulfhydryl groups present in the reaction. Reaction conditions and
results are found in Table 2; volumes are in μls, reaction 1 was
incubated for 5 min. at room temperature prior to absorbance reading, and
reactions 2-5 were incubated for 10 min. at room temperature prior to
absorbance readings.

As seen in Table 2, the positive control (reaction 1) and the test
reaction 5 (with amifostine) have similar absorbance readings, indicating
that the reaction conditions are capable of measuring free sulfhydryl
groups after dephosphorylation of a compound with alkaline phosphatase
(reaction 5).

[0075]A second assay was performed to examine the ability of alkaline
phosphatase to dephosphorylate compound (7) to create sulfhydryl reactive
groups. A 12.5 mM solution of Compound 7 was made (4 mg/ml) and used in
the test reactions. Reaction conditions and results are found in Table 3;
volumes are in μls, reactions were incubated for 10 min. at 37°
C. prior to absorbance readings, duplicates of the Compound 7 (C7)
negative reaction (without CIAP; reactions 6 & 8) and Compound 7 test
reaction (with CIAP; reactions 7 & 9) were performed.

As seen in Table 3, Compound 7 is dephosphorylated by alkaline phosphatase
to yield free reactive sulfhydryl groups. Such reactive sulfhydryl groups
are capable of capturing free oxygen radicals created by chemotherapy
and/or radiation therapy, thereby inhibiting or decreasing toxicity of
these compounds to normal cells and tissues. A time course of
dephosphorylation was also performed using Compound 7, following the same
reaction conditions as in Table 3. The time course showed that over a 30
min. period (A412 readings taken at 3 min. intervals) the
dephosphorylation of Compound 7 was time dependent, as an increase in
free sulfhydryl groups was seen over time.

Example 3

Intracellular Activity

[0076]The compound
(2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt) was tested for intracellular
properties. In particular, experiments were conducted to determine the
ability of the compound to enter into cells and generate glutathione.
HepG2 were incubated with the compound with or without added bovine
intestinal alkaline phosphatase (Sigma). Cells were scraped into SSA,
vortexed and then spun. GSH in the supernatants were analyzed utilizing
the glutathione reductase method of Tietze (Tietze F: "ENZYMIC METHOD FOR
QUANTITATIVE DETERMINATION OF NANOGRAM AMOUNTS OF TOTAL AND OXIDIZED
GLUTATHIONE APPLICATIONS TO MAMMALIAN BLOOD AND OTHER TISSUES" Analytical
Biochemistry, 27(3): 502-522 (1969)). The compound did not enter cells
unless the phosphate group was first hydrolyzed with alkaline
phosphatase. Cells treated with the compound and alkaline phosphatase had
a 3.6 fold increase in their GSH contents. Importantly, this increase in
cellular GSH levels also occurred in the presence of buthionine
sulfoximine (greater than 5 fold increase in cellular GSH), indicating
that the compound was not simply delivering cysteine or other building
blocks for GSH synthesis but rather delivering gamma-glutamyl cysteine.
Cells incubated with compound with or without alkaline phosphatase did
not exhibit any evidence of toxicity.

[0077]In experiments with mice and hamsters, no overt toxicity was
observed, with testing conducted at doses up to 5 mmoles/animal.

L-Glutamine (500 g, 3.42 mol) was stirred with 1M sodium bicarbonate
(10.26 L) and toluene (2.75 L). Benzyl chloroformate (684 ml, 818 g, 4.8
mol, 1.4 equiv.) was added dropwise over 60 min. and the resulting
mixture was stirred under nitrogen at room temperature overnight. Ethyl
acetate (6 L) was added, phases were separated. The organic phase was
extracted with water (1 L) and discarded. The aqueous phase was made
acidic with 6N hydrochloric acid (11.6 L) and extracted with ethyl
acetate (3×6 L). The combined extracts were washed with water (2
L), brine (2 L) and dried over sodium sulfate. After filtration, the
filtrate was concentrated in vacuo to give a residue which was triturated
with MTBE. The solid was filtered and was dried in a vacuum oven
(45° C.) to yield (823.4 g, 86%) of a solid.

[0079]MS (ESP): 303.0 (M+Na.sup.+) for C13H16N2O5

A mixture (S)-5-amino-2-(benzyloxycarbonylamino)-5-oxopentanoic acid of
(823 g, 2.94 mol), dimethylformamide (3 L) and sodium bicarbonate (1.481
Kg, 17.6 mol, 6 equiv.) was stirred at room temperature for 60 min. Ethyl
iodide (447 ml, 871 g, 5.6 mol, 1.9 equiv.) was added dropwise over 60
min. and stirring was continued for 4 days under nitrogen. The reaction
mixture was slowly diluted with water (10 L) and stirred for 60 min. The
solid was collected by filtration, washed with water (8 L) and dried in a
convection oven (50° C.) for 4 days to yield (905 g, 100%) of a
solid.

[0080]MS (ESP): 331.2 (M+Na.sup.+) for C15H20N2O5

A suspension of the (S)-ethyl
5-amino-2-(benzyloxycarbonylamino)-5-oxopentanoate (570 g, 1.85 mol) in
anhydrous acetonitrile (6 L) was stirred at reflux under nitrogen and
t-butyl nitrite (650 mL, 3.0 equiv.) was added quickly. The reflux was
continued for 2 hrs. After cooling, the solvent was removed in a rotary
evaporator. The residue was taken in water (1.5 L) and ethyl acetate (3
L) and the biphasic mixture was stirred well while solid sodium
bicarbonate was slowly added to pH=7.5. Phases were separated and the
organic phase was washed with 10% sodium bicarbonate (6×500 ml).
The combined aqueous extracts were washed with ethyl acetate (1 L), made
acidic with 6N hydrochloric acid and extracted with ethyl acetate
(4×750 ml). The combined extracts were dried over sodium sulfate
and concentrated in vacuo to yield (398 g, 70%) of a solid.

[0081]MS (ESP): 332.0 (M+Na.sup.+) for C15H19NO6

A suspension of (S)-4-(benzyloxycarbonylamino)-5-ethoxy-5-oxopentanoic
acid (398 g, 1.29 mol) in dry acetonitrile (4 L) was stirred under
nitrogen at room temperature and HOBt (209 g, 1.54 mol, 1.2 equiv.) was
added. Stirring was continued for 10 min, then EDCI (220 g, 1.42 mol, 1.1
equiv.) was added. The resulting mixture was stirred for 1.5 hrs when
serine ethyl ester free base (171 g, 1.29 mol, 1 equiv.) in acetonitrile
(1 L) was added. Stirring was continued at room temperature for 16 hrs.
The solvent was removed in vacuo and the residue was partitioned between
water (4 L) and ethyl acetate (8 L) and phases were separated. The
organic phase was washed successively with 5% potassium carbonate
(2×2 L) and brine (2×2 L), dried over sodium sulfate and the
solvent was removed in vacuo. The residue was triturated with MTBE,
filtered and dried in a vacuum oven (45° C.) to yield (381.4 g,
70%) as a solid.

[0082]MS (ESP): 447.0 (M+Na.sup.+) for C20H28N2O8

A solution of (S)-ethyl
2-(benzyloxycarbonylamino)-5-((S)-1-ethoxy-3-hydroxy-1-oxopropan-2-ylamin-
o)-5-oxopentanoate (381 g, 0.90 mol) in ethanol (7.5 L) containing 76 g of
10% palladium on activated carbon (50% water wet) was hydrogenated at 30
psi for 3 hrs. The catalyst was removed by filtration washing the cake
with ethanol (4×2 L). The solvent was removed in vacuo and the
residue was triturated with MTBE (2 L), filtered and dried in a vacuum
oven (45° C.) to yield (235.3 g, 91%) of a tan solid.

[0083]MS (ESP): 313.2 (M+Na.sup.+) for C12H22N2O6

A solution of (S)-ethyl
2-amino-5-((S)-1-ethoxy-3-hydroxy-1-oxopropan-2-ylamino)-5-oxopentanoate
(10 g, 35 mmol) in dichloromethane (350 ml) was treated with thionyl
chloride (20 mL) and stirred at room temperature under nitrogen
overnight. The solvent was removed on a rotary evaporator (bath
temperature below 28° C.). Dichloromethane (100 ml) was added and
stripped under the same conditions twice. The solid residue was
triturated with DCM (100 mL), Heptane (100 mL), and MTBE (100 ml),
filtered and dried in a vacuum oven (25° C.) to yield (10 g, 84%)
of an off-white solid.

[0084]MS (ESP): 309.0 (M+H.sup.+) for C12H21ClN2O5

To solution of 40 g of sodium hydroxide in 300 mL of water was added
thiophosphoryl chloride (28.6 g, 0.17 mol) in one portion and the
resulting biphasic solution is quickly heated to reflux. The reaction
mixture is heated at reflux until the thiophosphoryl chloride layer is no
longer observed (approx. 30 min.). The heating mantle was removed and the
reaction mixture cooled to room temperature. An ice water bath is used to
precipitate out the product and sodium salts (approx. 30 minutes at
0° C.). The mixture of product and sodium chloride are filtered
off, the solids are collected and dissolved in 150 mL of 45° C.
water (removes sodium chloride). Anhydrous methanol (200 mL) is added to
precipitate the product which is filtered, collected and stirred under
200 mL of anhydrous methanol for 16 hours to effectively dehydrate the
salt. The solids are again collected by filtration and dried in a vacuum
oven with no heat for 32 hours to yield (17.3 g, 56.5%) of a white solid.

To a 500 mL round bottom flask was added 250 mL DIUF water. Water was then
degassed with nitrogen over 20 min.
(S)-5-((R)-3-chloro-1-ethoxy-1-oxopropan-2-ylamino)-1-ethoxy-1,5-dioxopen-
tan-2-aminium chloride (5 g, 14.5 mmol) and freshly prepared
trisodiumthiophosphate (2.9 g, 16.0 mmol) were added at once. The
reaction mixture was stirred at room temperature under nitrogen for 3
days. The aqueous mixture was concentrated to a minimal volume in vacuo
keeping the bath temperature below 25° C. The aqueous residue (50
ml/run) was loaded onto an Analogix 300 g flash C18 column using water as
the eluent to yield (6.0 g) of a light yellow foamy solid that is very
hygroscopic.

In some embodiments, the following steps are used for producing compound 7
from compound 5.Synthesis of
3-(2-chloro-1-ethoxycarbonyl-ethylcarbamoyl)-1-ethoxycarbonyl-propyl-ammo-
nium chloride (6): A solution of alcohol 5 (10 g, 35 mmol) in
dichloromethane (350 ml) was treated with thionyl chloride (20 mL) and
stirred at room temperature under nitrogen overnight. The solvent was
removed on a rotary evaporator (bath temperature below 28° C.).
Dichloromethane (100 ml) was added and stripped under the same conditions
twice. The solid residue was triturated with DCM (100 mL), Heptane (100
mL), and MTBE (100 ml) to give 10 g (84% yield) of pure 6 (LCMS) as a
off-white solid.Synthesis of Trisodiumthiophosphate: To a flask was
charged 40 g (1.0 mol) of sodium hydroxide in 300 mL of water. The
solution is stirred until all of the base is dissolved. Thiophosphoryl
chloride (28.6 g, 0.17 mol) is added in one portion and the resulting
bi-phasic solution is quickly heated to reflux. The reaction mixture is
heated at reflux until the thiophosphoryl chloride layer is no longer
observed (approx. 30 min). The heating mantle is removed and the reaction
mixture cooled to room temperature. An ice water bath is used to
precipitate out the product and sodium salts (approx. 30 minutes at
0° C.). The mixture of product and sodium chloride is filtered
off, the solids are collected and dissolved in 150 mL of 45° C.
water (removes sodium chloride). Anhydrous methanol (200 mL) is added to
precipitate out the trisodiumphosphoryl chloride. The product is
filtered, collected and stirred under 200 mL of anhydrous methanol for 16
hours to effectively dehydrate the salt. The solids are again collected
by filtration and dried in a vacuum oven with no heat for 32 hours. 17.3
g of product is obtained in 56.5% yield.Synthesis of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt (7): To a 500 mL round bottom flask was
added 250 mL DIUF water. Water was then degassed by nitrogen over 20 min.
5 g 6 and 2.9 g fresh made trisodiumthiophosphate were added at once. The
reaction mixture was stirred at room temperature under nitrogen for 3
days. LC/MS indicated that the major peak is product. Analogix 300 g
flash C18 column was then applied to purify the final product to give 6.0
g light yellow clear film.

[0087]All publications and patents mentioned in the present application
are herein incorporated by reference. Various modification and variation
of the described methods and compositions of the invention will be
apparent to those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be understood
that the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the described
modes for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the following
claims.